Effect of MMP12 transfection on in vitro and in vivo angiogenesis. ECFCs were transiently transfected with the recombinant vector pCDNA3.1 + MMP12. As control of transfection, empty vector pCDNA3.1 was used. (A) Left panel (top part): Western blotting analysis of MMP12 released in the medium from control and MMP12 transiently transfected ECFCs. Left panel (bottom part): truncation of uPAR in MMP12-transfected ECFCs. Numbers on the right show molecular weight expressed in kilodaltons. This figure shows typical results of 3 different experiments that gave similar results. The middle part of the figure shows Matrigel invasion (top panels) and capillary morphogenesis (bottom panels) of MMP12 transiently transfected ECFCs. Results are the mean of 3 different experiments performed in triplicate with 3 ECFC preparations. Histograms: Quantification of Matrigel invasion and capillary morphogenesis performed by counting migrated cells and percentage field occupancy of capillary projections, respectively. Image acquisition and quantification were performed as described in Figures 1 and 3. Results are shown as mean value ± SD. *P < .05, significantly different from control. (B) Western blotting showing MMP12-dependent uPAR cleavage. Standard uPAR (st) was incubated overnight with culture medium from control, empty vector and MMP12 transiently transfected ECFCs, in the absence or presence of anti-MMP12 antibody. st indicates standard uPAR incubated in EBM-2 medium with 2% FBS. Full-length and truncated forms of uPAR were evaluated by Western blotting analysis with the specific monoclonal antibody anti-uPAR M2. The figure shows the results of a typical experiment of 5 different experiments. (C) In vivo Matrigel sponge assay. Flanks of mice were injected with Matrigel (4 injections/animal) containing aliquots of 50 μL of reconstituted culture medium of ECFCs transfected with recombinant vector pCDNA3.1 + MMP12 or with pCDNA3.1 (empty vector), in the absence or in the presence of anti-MMP12 antibody (10 μg/mL), or of an irrelevant Ig. Top panels: Quantification of the experiment by evaluating hemoglobin (Hb) content of the sponges under the various experimental conditions (4 injections/animal; 2 animals for each condition). Graphs are shown as mean ± SD. *P < .05. Bottom panels: A representative photograph, taken with the Zeiss SR Stemi stereomicroscope, of the vascularization of individual Matrigel sponges recovered at autopsy for the corresponding condition and representative of a typical experiment. Image acquisition and quantification was performed as described in Figures 1 and 3. Inset (C top part): Western blotting of standard uPAR (st) incubated with aliquots of the solubilized plugs and then evaluated by Western blotting, using the M2 antibody (which recognizes both native and truncated uPAR forms). Only plug-extracted CM of pMMP12-ECFC cleaved uPAR. Such inhibition was counteracted by anti-MMP12 antibody but not by an irrelevant Ig, thus showing the specificity of anti-MMP12 antibodies. The figure shows a typical Western blotting pattern, of 5 experiments performed with different plugs extracts, that gave similar results.